Alfalfa Variety 09N12CY

ABSTRACT

A novel alfalfa variety designated 09N12CY and seed, plants and plant parts thereof. Methods for producing an alfalfa plant that comprise crossing alfalfa variety 09N12CY with another alfalfa plant. Methods for producing an alfalfa plant containing in its genetic material one or more traits introgressed into 09N12CY through backcross conversion and/or transformation, and to the alfalfa seed, plant and plant part produced thereby. Alfalfa seed, plant or plant part produced by crossing alfalfa variety 09N12CY or a trait conversion of 09N12CY with another alfalfa plant or population. Alfalfa populations derived from alfalfa variety 09N12CY, methods for producing other alfalfa populations derived from alfalfa variety 09N12CY and the alfalfa populations and their parts derived by the use of those methods.

FIELD OF INVENTION

This invention is in the field of alfalfa (Medicago sativa) breeding,specifically relating to an alfalfa variety designated 09N12CY.

BACKGROUND OF THE INVENTION

Alfalfa (Medicago sativa L., also known as lucerne) is one of theworld's most valuable forage legumes. It is grown for hay, pasture andsilage, and is valued highly as a livestock feed. Alfalfa is highlyeffective in nitrogen fixation, and is frequently planted in croprotation to replenish nutrients depleted from the soil by other cropssuch as corn.

Alfalfa originated in the Near East, in the area extending from Turkeyto Iran and north into the Caucasus. From the great diversity of formswithin the genus Medicago, two species, M. sativa and M. falcata, havebecome important forage plants. These species are mainly tetraploid,with 32 chromosomes, although diploid forms are known.

Alfalfa is a herbaceous perennial legume characterized by a deep taproot showing varying degrees of branching. Erect or semi-erect stems,bearing an abundance of leaves, grow to a height of 2-4 feet. The numberof stems arising from a single woody crown may vary from just a few to50 or more. New stems develop when older ones mature or have been cut orgrazed. Flowers are borne on axillary racemes which vary greatly in sizeand number of flowers. Flower color is predominantly purple, orbluish-purple, but other colors occur. The fruit is a legume, or pod,usually spirally coiled in M. sativa. Seeds are small, with about220,000/lb., and the color varies from yellow to brown. Alfalfa iswidely adapted to temperature and soil conditions, except for humidtropical conditions. Reproduction in alfalfa is mainly bycross-fertilization, but substantial self-pollination may also occur.Cross-pollination is effected largely by bees.

The commercial production of seeds for growing alfalfa plants normallyinvolves four stages, the production of breeder, foundation, certifiedand registered seeds. Breeder seed is the initial increase of seed ofthe strain which is developed by the breeder and from which foundationseed is derived. Foundation seed is the second generation of seedincrease and from which certified seed is derived. Certified seeds areused in commercial crop production and are produced from foundation orcertified seed. Foundation seed normally is distributed by growers orseedsmen as planting stock for the production of certified seed.

BRIEF SUMMARY OF THE INVENTION

According to the invention, there is provided a novel alfalfa variety,designated 09N12CY and processes for making 09N12CY. This inventionrelates to seed of alfalfa variety 09N12CY, to the plants of alfalfavariety 09N12CY, to plant parts of alfalfa variety 09N12CY, and toprocesses for making an alfalfa plant that comprise crossing alfalfavariety 09N12CY with another alfalfa plant. This invention also relatesto processes for making an alfalfa plant containing in its geneticmaterial one or more traits introgressed into 09N12CY through backcrossconversion and/or transformation, and to the alfalfa seed, plant andplant part produced by such introgression. This invention furtherrelates to alfalfa seed, plant or plant part produced by crossing thealfalfa variety 09N12CY or an introgressed trait conversion of 09N12CYwith another alfalfa population. This invention also relates to alfalfapopulations derived from alfalfa variety 09N12CY to processes for makingother alfalfa populations derived from alfalfa variety 09N12CY and tothe alfalfa populations and their parts derived by the use of thoseprocesses.

DETAILED DESCRIPTION OF THE INVENTION AND FURTHER EMBODIMENTSDefinitions

Acid-Detergent Fiber (“ADF”) approximates the amount of cellulose fiberand ash present in a feed. Forages with high ADF values are lessdigestible than forages with low ADF values and, therefore, providefewer nutrients to the animal through digestion. Because of thisrelationship, ADF serves as an estimate of digestibility and can be usedby nutritionists to predict the energy that will be available from aforage.

AOSCA. Abbreviation for Association of Official Seed CertifyingAgencies.

Crude Protein (“CP”) is determined by measuring the total nitrogenconcentration of a forage and multiplying it by 6.25. This techniquemeasures not only the nitrogen present in true proteins, but also thatpresent in non-protein forms such as ammonia, urea and nitrate. Becausemost of the non-protein forms of nitrogen are converted to true proteinby the rumen microorganisms, CP is considered by nutritionists toprovide an accurate measure of the protein that will be available toruminant animals from a given forage.

DM. Abbreviation for Dietary Dry Matter. Used to calculate yield.

Fall Dormancy (Dormancy or “FD”)—Most alfalfa plants go dormant in thefall in preparation for winter. The onset of dormancy is triggered by acombination of day length and temperature and is genotype dependent.Fall dormancy scores indicate the dormancy response of alfalfa genotypesby quantifying the height of alfalfa measured in October relative to aset of standard check varieties. The standard fall dormancy testrequires that plants are cut off in early September with plant heightmeasured in early-mid October. Early fall dormant types show very littlegrowth after the September clipping, later fall dormant type demonstratesubstantial growth.

Alfalfa is classified into fall dormancy groups, numbered 1 to 11, whereDormancy Group 1 is most dormant and suited for cold climates (suchvarieties would stop growing and go dormant over winter), and DormancyGroup 7-11 are very non-dormant and suited for very hot climates (suchvarieties would have high growth rates over a very long growing seasonand would have relatively high winter activity). The NA&MLVRB recognizesstandard or check varieties for Dormancy Groups 1-11, Check cultivarsare listed in the NAAIC Standard Tests to Characterize AlfalfaCultivars, maintained online on the NAAIC's website. The check varietiesfor the various fall dormancy ratings/Dormancy Groups (corresponding tothe rating scale used by the Certified Alfalfa Seed Council (CASC)) areas follows:

Check Cultivars

A single set of check cultivars representing fall dormancy classes (FDC)1 to 11 are designated. These check cultivars have been selected tomaintain the intended relationship between the original set of ninecheck cultivars (Standard Tests, March 1991, updated in 1998) and tohave minimal variation across environments. The actual fall dormancyrating (FDR) based on the average University of California regressionand the Certified Alfalfa Seed Council Class that each check cultivarrepresents are listed below.

Variety FDR¹ FDC² Maverick 0.8 1.0 Vernal 2.0 2.0 5246 3.4 3.0 Legend3.8 4.0 Archer 5.3 5.0 ABI 700 6.3 6.0 Doña Ana 6.7 7.0 Pierce 7.8 8.0CUF 101 8.9 9.0 UC-1887 9.9 10.0 UC-1465 11.2 11.0 The FDR¹ numbercorresponds to the value calculated using the University of Californiaregression equation. The FDC² number corresponds to fall dormancy classused by the Certified Alfalfa Seed Council (CASC)

In Vitro True Digestibility (“IVTD”) is a measurement of digestibilityutilizing actual rumen microorganisms. Although ADF serves as a goodestimate of digestibility, IVID provides a more accurate assessment of aforage's feeding value by actually measuring the portion of a foragethat is digested. This process is more expensive and time consuming thanthe analysis for ADF concentrations of a feed, but provides a moremeaningful measure of forage digestibility. Techniques for measuring invitro digestibility are based on incubating a forage sample in asolution containing rumen microorganisms for an extended period of time(usually 48 hours).

TA. Abbreviation for Tons per Acre. Used to calculate yield.

Milk Per Ton is an estimate of the milk production that could besupported by a given forage when fed as part of a total mixed ration.The equation for calculating milk per ton uses NDF and ADF to calculatetotal energy intake possible from the forage. After subtracting theamount of energy required for daily maintenance of the cow, the quantityof milk that could be produced from the remaining energy is calculated.The ratio of milk produced to forage consumed is then reported in theunits of pounds of milk produced per ton of forage consumed. Milk perton is useful because it characterizes forage quality in two terms thata dairy farmer is familiar with: pounds of milk and tons of forage. Bycombining milk per ton and dry matter yield per acre, we arrive at “milkper acre”. This term is widely used to estimate the economic value of aforage.

NAAIC. Abbreviation for North America Alfalfa Improvement Conference,which is the governing body over the NA&MLVRB

NA&MLVRB. Abbreviation for National Alfalfa and Miscellaneous LegumeVariety Review Board. The NA&MLVRB is administered by the Association ofOfficial Seed Certifying Agencies (AOSCA).

NAVRB. Abbreviation for National Alfalfa Variety Review Board. NAVRBrecently changed its name to “National Alfalfa and Miscellaneous LegumeVariety Review Board” (NA&MLVRB).

Neutral-Detergent Fiber (“NDF”) represents the total amount of fiberpresent in the alfalfa. Because fiber is the portion of the plant mostslowly digested in the rumen, it is this fraction that fills the rumenand becomes a limit to the amount of feed an animal can consume. Thehigher the NDF concentration of a forage, the slower the rumen willempty reducing what an animal will be able to consume. For this reason,NDF is used by nutritionists as an estimate of the quantity of foragethat an animal will be able to consume. Forages with high NDF levels canlimit intake to the point that an animal is unable to consume enoughfeed to meet their energy and protein requirements.

Percentage of alfalfa plant having resistance to potatoleafhopper—Alfalfa varieties are heterogeneous populations formed byintercrossing a number of alfalfa clones. Pest resistance in alfalfavarieties is commonly measured in standard tests as the percent ofplants in the population that express the resistance trait. The NationalAlfalfa Variety Review Board in accordance with the recommendation ofthe North American Alfalfa Improvement Conference has adopted aconvention that uses percent resistant plants to describe levels of pestresistance. This convention is as follows: (0-5%)=susceptible,(6-15%)=low resistance, (16-30%)=moderate resistance,(31-50%)=resistance, and (>51%)=high resistance. With most pests,economic losses due to pest damage are minimized or eliminated withvarieties containing resistance to high resistance. Individual plantscan also have varying levels of resistance. The convention used formeasuring PLH damage in this application was patterned after standardtests used for measuring damage/resistance to other pests. Individualplants are scored on a (1-5) scale, where 1=no damage evident and5=severe stunting and yellowing. Plants scored as 1 and 2 are classifiedas resistant. The average severity index (ASI) of a variety is theaverage damage score for 100 random plants. The ASI is often used incombination with percent resistance to characterize pest resistance ofalfalfa cultivars.

Using this standard convention, an alfalfa variety described as beingresistant to PLH has between (31%-50%) of the plants in the varietybeing scored 1 or 2 in a standard test to measure PLH reaction.Individual alfalfa plants or clones (clonal propagules of individualgenotypes) with a resistance score of 1 have very high resistance; ascore of 3 show moderate resistance; and a score of 5 show noresistance.

Potato Leafhopper Resistance is a reaction of the alfalfa host plantwhich enables it to avoid serious damage from potato leafhopper feeding.The resistant plant reaction is to demonstrate normal growth in thepresence of high populations of potato leafhoppers, whereas susceptibleplants show significant stunting and yellowing in reaction to insectfeeding.

Relative Feed Value (“RFV”) is a numeric value assigned to forages basedupon their ADF and NDF values. In this calculation, NDF is used toestimate the dry matter intake expected for a given forage, and the ADFconcentration is used to estimate the digestibility of the forage. Bycombining these two relationships, an estimate of digestible dry matterintake is generated. This value is then reported relative to a standardforage (fall bloom alfalfa=100), and can be used to rank forages basedon their anticipated feeding value. Relative feed value has beenaccepted in many areas as a means of estimating forage feeding value andis commonly used in determining the price of alfalfa at tested hayauctions.

Relative Forage Quality (“RFQ”) is a numeric value that estimates theenergy content of forage for total digestible nutrients as recommendedby the National Research Council. Values are assigned to forages basedupon the actual fiber digestibility (NDFd) and Total DigestibleNutrients (TDN). By combining these two relationships, an estimate ofhow the forage will perform in animal rations is predicted. Relativeforage quality has been accepted in many areas as a means of estimatingforage feeding value and is commonly used in determining the price ofalfalfa at tested hay auctions or for on farm use.

Synthetic variety (“SYN”) is developed by intercrossing a number ofgenotypes with specific favorable characteristics and/or overall generalfavorable qualities. Synthetic (SYN) variety can be developed by usingclones, inbreds, open pollinated varieties, and/or individualheterozygous plants.

Total Digestible Nutrients (“TDN”) is an estimate of the energy contentof a feedstuff based on its relative proportions of fiber, fat,carbohydrate, crude protein, and ash. Because it is expensive to measureeach of these components, TDN is usually estimated from ADF or IVTD.Although still used in some areas as a criteria for evaluating alfalfahay at auctions, TDN has been shown to overestimate the energy contentof low quality forages and thus does not accurately reflect thenutritional value of all forage samples.

Winterhardiness (“WH”) is a measure of the ability of an alfalfa plantto survive the stresses associated with winter. Cold hardiness is a keyfeature of the winterhardiness trait. There is a general relationshipbetween fall dormancy and winterhardiness, the early fall dormant types(FD2-5) being more winterhardy than the later fall dormant types(FD6-9). The winterhardiness rating used in this patent are derived fromthe standard test for measuring winter survival. The standard testmeasures plant survival and spring vigor following a winter stressenough to substantially injure check varieties.

Morphological and Physiological Characteristics of Alfalfa Variety09N12CY

Alfalfa variety 09N12CY is an intercross of 22 clones selected forforage yield, persistence, forage quality, standability and orresistance to one or more of the following pests: bacterial wilt,Fusarium wilt, Verticillium wilt, anthracnose (Race 1), Phytophthoraroot rot, Aphanomyces root rot (Race 1 & 2), and stem nematode. Parentclones were identified using phenotypic selection in selection nurseriesfor standability (lodging tolerance) forage quality, increased pectin,persistence, agronomic characteristics, and improved forage yield.

Alfalfa variety 09N12CY is adapted to the North Central, East Central,and Moderately Winterhardy Intermountain regions of the U.S. Thisvariety has been tested in Wisconsin, Washington, Minnesota, Michigan,and Pennsylvania, and is intended for use in the North Central, EastCentral, Moderately Winterhardy Intermountain, WinterhardyIntermountain, and Great Plains regions of the U.S. and Canada.

09N12CY is Moderately Dormant, similar to FD5 check. Flower color (Syn2) is 94% purple, 3% cream, 3% variegated with a trace of yellow andwhite.

09N12CY is highly resistant to Aphanomyces root rot (Race 1), bacterialwilt, Verticillium wilt, Fusarium wilt, and Phytophthora root rot; withresistance to Anthracnose (Race 1), pea aphid, Aphanomyces root rot(Race 2), stem nematode, lodging and spotted alfalfa aphid.

Use of 09N12CY in Alfalfa Breeding

Alfalfa is an auto-tetraploid and is frequently self-incompatible inbreeding. When selfed, little or no seed is produced, or the seed maynot germinate, or when it does, may have reduced vigor and may laterstop growing. Typically, fewer than five percent of selfed crossesproduce seed. When a very small population is crossbred, inbreedingdepression occurs, and traits of interest, such as quality, yield, andresistance to a large number of pests (e.g., seven or eight differentpests), are lost. Thus, producing a true breeding parent for hybrids isnot possible, which complicates breeding substantially.

Efforts to develop alfalfa varieties having improved traits andincreased production have focused on breeding for disease, insect, ornematode resistance, persistence, adaptation to specific environments,increased yield, and improved quality. Breeders have had some success inbreeding for increased herbage quality and forage yield, although thereare significant challenges.

Breeding programs typically emphasize maximizing heterogeneity of agiven alfalfa variety to improve yield and stability. However, thisgenerally results in wide variations in characteristics such asflowering dates, flowering frequency, development rate, growth rate,fall dormancy and winter hardiness. Prior art breeding methods do notemphasize improving the uniformity of these characteristics.

Some sources indicate that there are nine major germplasm sources ofalfalfa: M. falcata, Ladak, M. varia, Turkistan, Flemish, Chilean,Peruvian, Indian, and African. Tissue culture of explant source tissue,such as mature cotyledons and hypocotyls, demonstrates the regenerationfrequency of genotypes in most cultivars is only about 10 percent.Seitz-Kris, M. H. and E. T. Bingham, In vitro Cellular and DevelopmentalBiology 24 (10):1047-1052 (1988). Efforts have been underway to improveregeneration of alfalfa plants from callus tissue. E. T. Bingham, et.al., Crop Science 15:719-721 (1975).

Another aspect of the present invention provides a method for producingfirst-generation synthetic variety alfalfa seed comprising crossing afirst parent alfalfa plant with a second parent alfalfa plant andharvesting resultant first-generation (F1) alfalfa seed, wherein saidfirst or second parent alfalfa plant is one of the alfalfa plants of thepresent invention described above.

There is a need in the art for producing alfalfa having agronomicallydesirable traits and breeding methods that result in a high degree ofhybridity, uniformity of selected traits, and acceptable seed yields.

Male Sterility

The present invention also provides a method of obtaining alfalfapopulations using cytoplasmic male sterile alfalfa populations (Apopulations), maintainer alfalfa populations (B populations), and malefertile pollenizer populations (C populations) as described in detail inthe examples.

Male sterile A populations may be identified by evaluating pollenproduction using the Pollen Production Index (P.P.I.), which recognizesfour distinct classes:

-   -   1. Male Sterile Plants (MS) PPI=0    -   No visible pollen can be observed with the naked eye when flower        is tripped with a black knife blade.    -   2. Partial Male Sterile Plant (PMS) PPI=0.1    -   A trace of pollen is found with the naked eye when flower is        tripped with a black knife blade.    -   3. Partial Fertile Plant (PF) PPI=0.6    -   Less than a normal amount of pollen can be observed with the        naked eye when flower is tripped with a black knife blade.    -   4. Fertile Plant (F) PPI=1.0    -   Normal amounts of pollen can be observed when flower is tripped        with a black knife blade.

The cells of the cytoplasmic male sterile (A population) alfalfa plantscontain sterile cytoplasm and the non-restorer gene. The maintainerpopulation (B population) is a male and female fertile plant, and whencrossed with an A population plant, maintains the male sterility of thecytoplasmic male sterile plant in the progeny. The cells of a maintainerpopulation plant contain normal cytoplasm and the non-restorer gene.Methods for identifying cytoplasmic male sterile and maintainerpopulations of alfalfa are well known to those versed in the art ofalfalfa plant breeding (e.g., see U.S. Pat. No. 3,570,181, which isincorporated by reference herein). A pollenizer population (Cpopulation) is a fertile plant containing both male and female parts.

Cytoplasmic male sterile populations may be maintained by vegetativecuttings. Maintainer populations can be maintained by cuttings orself-pollination. Male sterile plants can be obtained bycross-pollinating cytoplasmic male sterile plants with maintainerplants. Pollenizer populations can be maintained by selfing or, if morethan two clones are used, by cross-pollination.

Preferably, at least one of the alfalfa plant populations used indeveloping alfalfa plants according to the method of the presentinvention has at least one desirable agronomic trait, which may include,for example, resistance to disease or insects, cold tolerance, increasedpersistence, greater forage yield or seed yield, improved foragequality, uniformity of growth rate, and uniformity of time of maturity.

In the controlled pollination step, the cytoplasmic male sterile plantsare typically grown in separate rows from the maintainer plants. Theplants are pollinated by pollen-carrying insects, such as bees.Segregating the male sterile and maintainer plants facilitates selectiveharvest of seed from the cytoplasmic male sterile plants.

The male sterile seed and male fertile seed is preferably provided as arandom mixture of the seed in a ratio of about 4:1, which would providefor random distribution of the male sterile and male fertile plantsgrown therefrom and random pollination of the alfalfa plants. As one ofskill in the art will appreciate, one could also practice the method ofthe invention using designed distribution of male sterile and malefertile populations within a field and subsequent pollination bypollen-carrying insects.

One of ordinary skill in the art will appreciate that any suitable malesterile population, maintainer population, and pollenizer populationcould be successfully employed in the practice of the method of theinvention.

Tissue Culture

Yet another embodiment is a tissue culture of regenerable cells derived,in whole or in part, from an alfalfa plant of synthetic variety named09N12CY. In one such embodiment, the cells regenerate plants havingsubstantially all the morphological and physiological characteristics ofthe synthetic alfalfa variety named 09N12CY that are described in theattached tables. Some embodiments include such a tissue culture thatincludes cultured cells derived, in whole or in part, from a plant partselected from the group consisting of leaves, roots, root tips, roothairs, anthers, pistils, stamens, pollen, ovules, flowers, seeds,embryos, stems, buds, cotyledons, hypocotyls, cells and protoplasts.Another embodiment is an alfalfa plant regenerated from such a tissueculture, having all the morphological and physiological characteristicsof synthetic alfalfa variety 09N12CY.

Some methods for regeneration of alfalfa plants from tissue culture aredescribed in U.S. Pat. No. 5,324,646 issued Jun. 28, 1994, which ishereby incorporated by reference. Additionally, researchers believe thatsomatic embryogenesis in alfalfa is heritable, and is controlled byrelatively few genes. Efforts at improving regeneration have thus beendirected towards isolation of the genetic control of embryogenesis, andbreeding programs which would incorporate such information. See, e.g.,M. M. Hernandez-Fernandez, and B. R. Christie, Genome 32:318-321 (1989);I. M. Ray and E. T. Bingham, Crop Science 29:1545-1548 (1989).

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cells of tissue culture from which alfalfa plants canbe regenerated, plant calli, plant clumps, and plant cells that areintact in plants or parts of plants such as pollen, flowers, seeds,leaves, stems, and the like.

Tissue culture of alfalfa is further described in Saunders, J. W. andBingham, E. T., (1971) Production of alfalfa plants from callus tissue,Crop Sci 12; 804-808, and incorporated herein by reference.

Transformation

The advent of new molecular biological techniques has allowed theisolation and characterization of genetic elements with specificfunctions, such as encoding specific protein products. Scientists in thefield of plant biology developed a strong interest in engineering thegenome of plants to contain and express foreign genetic elements, oradditional, or modified versions of native or endogenous geneticelements in order to alter the traits of a plant in a specific manner.Any DNA sequences, whether from a different species or from the samespecies, which are inserted into the genome using transformation arereferred to herein collectively as “transgenes”. In some embodiments ofthe invention, a transformed variant of 09N12CY may contain at least onetransgene but could contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10and/or no more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2.Over the last fifteen to twenty years several methods for producingtransgenic plants have been developed, and the present invention alsorelates to transformed versions of the claimed alfalfa variety 09N12CYas well as hybrid combinations thereof.

Numerous methods for plant transformation have been developed, includingbiological and physical plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glick,B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88 and Armstrong, “The First Decade of Maize Transformation: A Reviewand Future Perspective” (Maydica 44:101-109, 1999). Specific to alfalfa,see “Efficient Agrobacterium-mediated transformation of alfalfa usingsecondary somatic embryogenic callus”, Journal of the Korean Society ofGrassland Science 20 (1): 13-18 2000, E. Charles Brummer, “ApplyingGenomics to Alfalfa Breeding Programs” Crop Sci. 44:1904-1907 (2004),and “Genetic transformation of commercial breeding populations ofalfalfa (Medicago sativa)” Plant Cell Tissue and Organ Culture 42 (2):129-140 1995 which are incorporated by reference for this purpose. Inaddition, expression vectors and in vitro culture methods for plant cellor tissue transformation and regeneration of plants are available. See,for example, Gruber et al., “Vectors for Plant Transformation” inMethods in Plant Molecular Biology and Biotechnology, Glick, B. R. andThompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages 89-119.

The most prevalent types of plant transformation involve theconstruction of an expression vector. Such a vector comprises a DNAsequence that contains a gene under the control of or operatively linkedto a regulatory element, for example a promoter. The vector may containone or more genes and one or more regulatory elements.

A genetic trait which has been engineered into the genome of aparticular alfalfa plant using transformation techniques, could be movedinto the genome of another population using traditional breedingtechniques that are well known in the plant breeding arts. For example,a backcrossing approach may be used to move a transgene from atransformed alfalfa plant to an elite population, and the resultingprogeny would then comprise the transgene(s).

Various genetic elements can be introduced into the plant genome usingtransformation. These elements include, but are not limited to genes;coding sequences; inducible, constitutive, and tissue specificpromoters; enhancing sequences; and signal and targeting sequences. Forexample, see the traits, genes and transformation methods listed in U.S.Pat. No. 6,118,055.

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants that areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114: 92-6(1981).

A genetic map can be generated, primarily via conventional RestrictionFragment Length Polymorphisms (RFLP), Polymerase Chain Reaction (PCR)analysis, Simple Sequence Repeats (SSR) and Single NucleotidePolymorphisms (SNP) that identifies the approximate chromosomal locationof the integrated DNA molecule. For exemplary methodologies in thisregard, see Glick and Thompson, Methods in Plant Molecular Biology andBiotechnology 269-284 (CRC Press, Boca Raton, 1993). Specific toalfalfa, see Construction of an improved linkage map of diploid alfalfa(Medicago sativa), Theoretical and Applied Genetics 100 (5): 641-657March, 2000 and Isolation of a full-length mitotic cyclin cDNA cloneCycIIIMs from Medicago sativa: Chromosomal mapping and expression, PlantMolecular Biology 27 (6): 1059-1070 1995 which are incorporated byreference for this purpose.

Wang et al. discuss “Large Scale Identification, Mapping and Genotypingof Single-Nucleotide Polymorphisms in the Human Genome”, Science,280:1077-1082, 1998, and similar capabilities are becoming increasinglyavailable for many plant genomes. Map information concerning chromosomallocation is useful for proprietary protection of a subject transgenicplant. If unauthorized propagation is undertaken and crosses made withother germplasm, the map of the integration region can be compared tosimilar maps for suspect plants to determine if the latter have a commonparentage with the subject plant. Map comparisons would involvehybridizations, RFLP, PCR, SSR and sequencing, all of which areconventional techniques. SNPs may also be used alone or in combinationwith other techniques.

Likewise, by means of the present invention, plants can be geneticallyengineered to express various phenotypes of agronomic interest. Throughthe transformation of alfalfa the expression of genes can be altered toenhance disease resistance, insect resistance, herbicide resistance,agronomic, grain quality and other traits. Transformation can also beused to insert DNA sequences which control or help controlmale-sterility. DNA sequences native to alfalfa as well as non-nativeDNA sequences can be transformed into alfalfa and used to alter levelsof native or non-native proteins. Various promoters, targetingsequences, enhancing sequences, and other DNA sequences can be insertedinto the alfalfa genome for the purpose of altering the expression ofproteins. Reduction of the activity of specific genes (also known asgene silencing, or gene suppression) is desirable for several aspects ofgenetic engineering in plants.

Many techniques for gene silencing are well known to one of skill in theart, including but not limited to knock-outs (such as by insertion of atransposable element such as mu (Vicki Chandler, The Maize Handbook ch.118 (Springer-Verlag 1994) or other genetic elements such as a FRT, Loxor other site specific integration site, antisense technology (see,e.g., Sheehy et al. (1988) PNAS USA 85:8805-8809; and U.S. Pat. Nos.5,107,065; 5,453, 566; and 5,759,829); co-suppression (e.g., Taylor(1997) Plant Cell 9:1245; Jorgensen (1990) Trends Biotech.8(12):340-344; Flavell (1994) PNAS USA 91:3490-3496; Finnegan et al.(1994) Bio/Technology 12: 883-888; and Neuhuber et al. (1994) Mol. Gen.Genet. 244:230-241); RNA interference (Napoli et al. (1990) Plant Cell2:279-289; U.S. Pat. No. 5,034,323; Sharp (1999) Genes Dev. 13:139-141;Zamore et al. (2000) Cell 101:25-33; and Montgomery et al. (1998) PNASUSA 95:15502-15507), virus-induced gene silencing (Burton, et al. (2000)Plant Cell 12:691-705; and Baulcombe (1999) Curr. Op. Plant Bio.2:109-113); target-RNA-specific ribozymes (Haseloff et al. (1988) Nature334: 585-591); hairpin structures (Smith et al. (2000) Nature407:319-320; WO 99/53050; and WO 98/53083); MicroRNA (Aukerman & Sakai(2003) Plant Cell 15:2730-2741); ribozymes (Steinecke et al. (1992) EMBOJ. 11:1525; and Perriman et al. (1993) Antisense Res. Dev. 3:253);oligonucleotide mediated targeted modification (e.g., WO 03/076574 andWO 99/25853); Zn-finger targeted molecules (e.g., WO 01/52620; WO03/048345; and WO 00/42219); and other methods or combinations of theabove methods known to those of skill in the art.

Exemplary nucleotide sequences that may be altered by geneticengineering include, but are not limited to, those categorized below.

1. Transgenes That Confer Resistance To Insects Or Disease And ThatEncode:

(A) Plant disease resistance genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266: 789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262: 1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinoset al., Cell 78: 1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae); McDowell & Woffenden, (2003) Trends Biotechnol.21(4): 178-83 and Toyoda et al., (2002) Transgenic Res. 11(6):567-82. Aplant resistant to a disease is one that is more resistant to a pathogenas compared to the wild type plant.

(B) A Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon. See, for example, Geiser et al.,Gene 48: 109 (1986), who disclose the cloning and nucleotide sequence ofa Bt delta-endotoxin gene. Moreover, DNA molecules encodingdelta-endotoxin genes can be purchased from American Type CultureCollection (Rockville, Md.), for example, under ATCC Accession Nos.40098, 67136, 31995 and 31998. Other examples of Bacillus thuringiensistransgenes being genetically engineered are given in the followingpatents and patent applications and hereby are incorporated by referencefor this purpose: 5,188,960; 5,689,052; 5,880,275; WO 91/14778; WO99/31248; WO 01/12731; WO 99/24581; WO 97/40162 and U.S. applicationSer. Nos. 10/032,717; 10/414,637; and 10/606,320.

(C) An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344: 458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

(D) An insect-specific peptide which, upon expression, disrupts thephysiology of the affected pest. For example, see the disclosures ofRegan, J. Biol. Chem. 269: 9 (1994) (expression cloning yields DNAcoding for insect diuretic hormone receptor); Pratt et al., Biochem.Biophys. Res. Comm. 163: 1243 (1989) (an allostatin is identified inDiploptera puntata); Chattopadhyay et al. (2004) Critical Reviews inMicrobiology 30 (1): 33-54 2004; Zjawiony (2004) J Nat Prod 67 (2):300-310; Carlini & Grossi-de-Sa (2002) Toxicon, 40 (11): 1515-1539;Ussuf et al. (2001) Curr Sci. 80 (7): 847-853; and Vasconcelos &Oliveira (2004) Toxicon 44 (4): 385-403. See also U.S. Pat. No.5,266,317 to Tomalski et al., who disclose genes encodinginsect-specific toxins.

(E) An enzyme responsible for a hyperaccumulation of a monterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

(F) An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23: 691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21: 673 (1993), who provide the nucleotide sequenceof the parsley ubi4-2 polyubiquitin gene, U.S. application Ser. Nos.10/389,432, 10/692,367, and U.S. Pat. No. 6,563,020.

(G) A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24: 757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104: 1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

(H) A hydrophobic moment peptide. See PCT application WO 95/16776 andU.S. Pat. No. 5,580,852 (disclosure of peptide derivatives ofTachyplesin which inhibit fungal plant pathogens) and PCT application WO95/18855 and U.S. Pat. No. 5,607,914) (teaches synthetic antimicrobialpeptides that confer disease resistance).

(I) A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure by Jaynes et al., Plant Sci. 89: 43 (1993),of heterologous expression of a cecropin-beta lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

(J) A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. Rev. Phytopathol.28: 451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

(K) An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor et al., Abstract #497, SEVENTH INT'L SYMPOSIUM ON MOLECULARPLANT-MICROBE INTERACTIONS (Edinburgh, Scotland, 1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

(L) A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366: 469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

(M) A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo alpha-1,4-D-polygalacturonasesfacilitate fungal colonization and plant nutrient release bysolubilizing plant cell wall homo-alpha-1,4-D-galacturonase. See Lamb etal., Bio/Technology 10: 1436 (1992). The cloning and characterization ofa gene which encodes a bean endopolygalacturonase-inhibiting protein isdescribed by Toubart et al., Plant J. 2: 367 (1992).

(N) A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology 10: 305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

(O) Genes involved in the Systemic Acquired Resistance (SAR) Responseand/or the pathogenesis related genes. Briggs, S., Current Biology,5(2):128-131 (1995), Pieterse & Van Loon (2004) Curr. Opin. Plant Bio.7(4):456-64 and Somssich (2003) Cell 113(7):815-6.

(P) Antifungal genes (Cornelissen and Melchers, Pl. Physiol.101:709-712, (1993) and Parijs et al., Planta 183:258-264, (1991) andBushnell et al., Can. J. of Plant Path. 20(2):137-149 (1998). Also seeU.S. application Ser. No. 09/950,933.

(Q) Detoxification genes, such as for fumonisin, beauvericin,moniliformin and zearalenone and their structurally related derivatives.For example, see U.S. Pat. No. 5,792,931.

(R) Cystatin and cysteine proteinase inhibitors. See U.S. applicationSer. No. 10/947,979.

(S) Defensin genes. See WO03000863 and U.S. application Ser. No.10/178,213.

(T) Genes conferring resistance to nematodes. See WO 03/033651 and Urwinet. al., Planta 204:472-479 (1998), Williamson (1999) Curr Opin PlantBio. 2(4):327-31.

2. Transgenes That Confer Resistance To A Herbicide, For Example:

(A) A herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7: 1241 (1988), and Miki et al., Theor. Appl. Genet. 80: 449(1990), respectively. See also, U.S. Pat. Nos. 5,605,011; 5,013,659;5,141,870; 5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107;5,928,937; and 5,378,824; and international publication WO 96/33270,which are incorporated herein by reference for this purpose.

(B) Glyphosate (resistance imparted by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus phosphinothricin acetyl transferase (bar) genes), andpyridinoxy or phenoxy proprionic acids and cycloshexones (ACCaseinhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 toShah et al., which discloses the nucleotide sequence of a form of EPSPSwhich can confer glyphosate resistance. U.S. Pat. No. 5,627,061 to Barryet al. also describes genes encoding EPSPS enzymes. See also U.S. Pat.Nos. 6,566,587; 6,338,961; 6,248,876 B1; 6,040,497; 5,804,425;5,633,435; 5,145,783; 4,971,908; 5,312,910; 5,188,642; 4,940,835;5,866,775; 6,225,114 B1; 6,130,366; 5,310,667; 4,535,060; 4,769,061;5,633,448; 5,510,471; Re. 36,449; RE 37,287 E; and 5,491,288; andinternational publications EP1173580; WO 01/66704; EP1173581 andEP1173582, which are incorporated herein by reference for this purpose.Glyphosate resistance is also imparted to plants that express a genethat encodes a glyphosate oxido-reductase enzyme as described more fullyin U.S. Pat. Nos. 5,776,760 and 5,463,175, which are incorporated hereinby reference for this purpose. In addition glyphosate resistance can beimparted to plants by the over expression of genes encoding glyphosateN-acetyltransferase. See, for example, U.S. Application Ser. Nos.01/46227; 10/427,692 and 10/427,692. A DNA molecule encoding a mutantaroA gene can be obtained under ATCC accession No. 39256, and thenucleotide sequence of the mutant gene is disclosed in U.S. Pat. No.4,769,061 to Comai. European Patent Application No. 0 333 033 to Kumadaet al. and U.S. Pat. No. 4,975,374 to Goodman et al. disclose nucleotidesequences of glutamine synthetase genes which confer resistance toherbicides such as L-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in European PatentNo. 0 242 246 and 0 242 236 to Leemans et al. De Greef et al.,Bio/Technology 7: 61 (1989), describe the production of transgenicplants that express chimeric bar genes coding for phosphinothricinacetyl transferase activity. See also, U.S. Pat. Nos. 5,969,213;5,489,520; 5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477;5,646,024; 6,177,616 B1; and 5,879,903, which are incorporated herein byreference for this purpose. Exemplary genes conferring resistance tophenoxy proprionic acids and cycloshexones, such as sethoxydim andhaloxyfop, are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described byMarshall et al., Theor. Appl. Genet. 83: 435 (1992).

(C) A herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3: 169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441 and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J. 285:173 (1992).

(D) Acetohydroxy acid synthase, which has been found to make plants thatexpress this enzyme resistant to multiple types of herbicides, has beenintroduced into a variety of plants (see, e.g., Hattori et al. (1995)Mol Gen Genet. 246:419). Other genes that confer resistance toherbicides include: a gene encoding a chimeric protein of rat cytochromeP4507A1 and yeast NADPH-cytochrome P450 oxidoreductase (Shiota et al.(1994) Plant Physiol. 106:17), genes for glutathione reductase andsuperoxide dismutase (Aono et al. (1995) Plant Cell Physiol 36:1687, andgenes for various phosphotransferases (Datta et al. (1992) Plant MolBiol 20:619).

(E) Protoporphyrinogen oxidase (protox) is necessary for the productionof chlorophyll, which is necessary for all plant survival. The protoxenzyme serves as the target for a variety of herbicidal compounds. Theseherbicides also inhibit growth of all the different species of plantspresent, causing their total destruction. The development of plantscontaining altered protox activity which are resistant to theseherbicides are described in U.S. Pat. Nos. 6,288,306 B1; 6,282,837 B1;and 5,767,373; and international publication WO 01/12825.

3. Transgenes That Confer Or Contribute To an Altered GrainCharacteristic, Such As:

(A) Altered fatty acids, for example, by

-   -   (1) Down-regulation of stearoyl-ACP desaturase to increase        stearic acid content of the plant. See Knultzon et al., Proc.        Natl. Acad. Sci. USA 89: 2624 (1992) and WO99/64579 (Genes for        Desaturases to Alter Lipid Profiles in Corn),    -   (2) Elevating oleic acid via FAD-2 gene modification and/or        decreasing linolenic acid via FAD-3 gene modification (see U.S.        Pat. Nos. 6,063,947; 6,323,392; 6,372,965 and WO 93/11245),    -   (3) Altering conjugated linolenic or linoleic acid content, such        as in WO 01/12800,    -   (4) Altering LEC1, AGP, Dek1, Superal1, mi1ps, various Ipa genes        such as Ipa1, Ipa3, hpt or hggt. For example, see WO 02/42424,        WO 98/22604, WO 03/011015, U.S. Pat. No. 6,423,886, U.S. Pat.        No. 6,197,561, U.S. Pat. No. 6,825,397, US2003/0079247,        US2003/0204870, WO02/057439, WO03/011015 and Rivera-Madrid, R.        et. al. Proc. Natl. Acad. Sci. 92:5620-5624 (1995).

(B) Altered phosphorus content, for example, by the

-   -   (1) Introduction of a phytase-encoding gene would enhance        breakdown of phytate, adding more free phosphate to the        transformed plant. For example, see Van Hartingsveldt et al.,        Gene 127: 87 (1993), for a disclosure of the nucleotide sequence        of an Aspergillus niger phytase gene.    -   (2) Up-regulation of a gene that reduces phytate content. In        alfalfa, this, for example, could be accomplished, by cloning        and then re-introducing DNA associated with one or more of the        alleles, such as the LPA alleles, identified in maize mutants        characterized by low levels of phytic acid, such as in Raboy et        al., Maydica 35: 383 (1990) and/or by altering inositol kinase        activity as in WO 02/059324, US2003/0009011, WO 03/027243,        US2003/0079247, WO 99/05298, US6197561, US6291224, US6391348,        WO2002/059324, US2003/0079247, Wo98/45448, WO99/55882,        WO01/04147.

(C) Altered carbohydrates effected, for example, by altering a gene foran enzyme that affects the branching pattern of starch or a genealtering thioredoxin (See U.S. Pat. No. 6,531,648). See Shiroza et al.,J. Bacteriol. 170: 810 (1988) (nucleotide sequence of Streptococcusmutans fructosyltransferase gene), Steinmetz et al., Mol. Gen. Genet.200: 220 (1985) (nucleotide sequence of Bacillus subtilis levansucrasegene), Pen et al., Bio/Technology 10: 292 (1992) (production oftransgenic plants that express Bacillus licheniformis alpha-amylase),Elliot et al., Plant Molec. Biol. 21: 515 (1993) (nucleotide sequencesof tomato invertase genes), Søgaard et al., J. Biol. Chem. 268: 22480(1993) (site-directed mutagenesis of barley alpha-amylase gene), andFisher et al., Plant Physiol. 102: 1045 (1993) (maize endosperm starchbranching enzyme II), WO 99/10498 (improved digestibility and/or starchextraction through modification of UDP-D-xylose 4-epimerase, Fragile 1and 2, Ref1, HCHL, C4H), U.S. Pat. No. 6,232,529 (method of producinghigh oil seed by modification of starch levels (AGP)). The fatty acidmodification genes mentioned above may also be used to affect starchcontent and/or composition through the interrelationship of the starchand oil pathways.

(D) Altered antioxidant content or composition, such as alteration oftocopherol or tocotrienols. For example, see U.S. Pat. No. 6,787,683,US2004/0034886 and WO 00/68393 involving the manipulation of antioxidantlevels through alteration of a phytl prenyl transferase (ppt), WO03/082899 through alteration of a homogentisate geranyl geranyltransferase (hggt).

(E) Altered essential seed amino acids. For example, see US6127600(method of increasing accumulation of essential amino acids in seeds),US6080913 (binary methods of increasing accumulation of essential aminoacids in seeds), US5990389 (high lysine), WO99/40209 (alteration ofamino acid compositions in seeds), WO99/29882 (methods for alteringamino acid content of proteins), US5850016 (alteration of amino acidcompositions in seeds), WO98/20133 (proteins with enhanced levels ofessential amino acids), US5885802 (high methionine), US5885801 (highthreonine), US6664445 (plant amino acid biosynthetic enzymes), US6459019(increased lysine and threonine), US6441274 (plant tryptophan synthasebeta subunit), US6346403 (methionine metabolic enzymes), US5939599 (highsulfur), US5912414 (increased methionine), WO98/56935 (plant amino acidbiosynthetic enzymes), WO98/45458 (engineered seed protein having higherpercentage of essential amino acids), WO98/42831 (increased lysine),US5633436 (increasing sulfur amino acid content), US5559223 (syntheticstorage proteins with defined structure containing programmable levelsof essential amino acids for improvement of the nutritional value ofplants), WO96/01905 (increased threonine), WO95/15392 (increasedlysine), US2003/0163838, US2003/0150014, US2004/0068767, US6803498,WO01/79516, and WO00/09706 (Ces A: cellulose synthase), U.S. Pat. No.6,194,638 (hemicellulose), U.S. Pat. No. 6,399,859 and US2004/0025203(UDPGdH), U.S. Pat. No. 6,194,638 (RGP).

4. Genes That Control Male-Sterility

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar et al. and chromosomal translocations asdescribed by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. Inaddition to these methods, Albertsen et al., U.S. Pat. No. 5,432,068,describe a system of nuclear male sterility which includes: identifyinga gene which is critical to male fertility; silencing this native genewhich is critical to male fertility; removing the native promoter fromthe essential male fertility gene and replacing it with an induciblepromoter; inserting this genetically engineered gene back into theplant; and thus creating a plant that is male sterile because theinducible promoter is not “on” resulting in the male fertility gene notbeing transcribed. Fertility is restored by inducing, or turning “on”,the promoter, which in turn allows the gene that confers male fertilityto be transcribed.

(A) Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN-Ac-PPT (WO 01/29237).

(B) Introduction of various stamen-specific promoters (WO 92/13956, WO92/13957).

(C) Introduction of the barnase and the barstar gene (Paul et al. PlantMol. Biol. 19:611-622, 1992).

For additional examples of nuclear male and female sterility systems andgenes, see also, U.S. Pat. No. 5,859,341; U.S. Pat. No. 6,297,426; U.S.Pat. No. 5,478,369; U.S. Pat. No. 5,824,524; U.S. Pat. No. 5,850,014;and U.S. Pat. No. 6,265,640; all of which are hereby incorporated byreference.

5. Genes that create a site for site specific DNA integration. Thisincludes the introduction of FRT sites that may be used in the FLP/FRTsystem and/or Lox sites that may be used in the Cre/Loxp system. Forexample, see Lyznik, et al., Site-Specific Recombination for GeneticEngineering in Plants, Plant Cell Rep (2003) 21:925-932 and WO 99/25821,which are hereby incorporated by reference. Other systems that may beused include the Gin recombinase of phage Mu (Maeser et al., 1991; VickiChandler, The Maize Handbook ch. 118 (Springer-Verlag 1994), the Pinrecombinase of E. coli (Enomoto et al., 1983), and the R/RS system ofthe pSR1 plasmid (Araki et al., 1992).6. Genes that affect abiotic stress resistance (including but notlimited to flowering, ear and seed development, enhancement of nitrogenutilization efficiency, altered nitrogen responsiveness, droughtresistance or tolerance, cold resistance or tolerance, and saltresistance or tolerance) and increased yield under stress. For example,see: WO 00/73475 where water use efficiency is altered throughalteration of malate; U.S. Pat. No. 5,892,009, U.S. Pat. No. 5,965,705,U.S. Pat. No. 5,929,305, U.S. Pat. No. 5,891,859, U.S. Pat. No.6,417,428, U.S. Pat. No. 6,664,446, U.S. Pat. No. 6,706,866, U.S. Pat.No. 6,717,034, U.S. Pat. No. 6,801,104, WO2000060089, WO2001026459,WO2001035725, WO2001034726, WO2001035727, WO2001036444, WO2001036597,WO2001036598, WO2002015675, WO2002017430, WO2002077185, WO2002079403,WO2003013227, WO2003013228, WO2003014327, WO2004031349, WO2004076638,WO9809521, and WO9938977 describing genes, including CBF genes andtranscription factors effective in mitigating the negative effects offreezing, high salinity, and drought on plants, as well as conferringother positive effects on plant phenotype; US2004/0148654 and WO01/36596where abscisic acid is altered in plants resulting in improved plantphenotype such as increased yield and/or increased tolerance to abioticstress; WO2000/006341, WO04/090143, U.S. application Ser. Nos.10/817,483 and 09/545,334 where cytokinin expression is modifiedresulting in plants with increased stress tolerance, such as droughttolerance, and/or increased yield. Also see WO0202776, WO2003052063,JP2002281975, U.S. Pat. No. 6,084,153, WO0164898, U.S. Pat. No.6,177,275, and U.S. Pat. No. 6,107,547 (enhancement of nitrogenutilization and altered nitrogen responsiveness). For ethylenealteration, see US20040128719, US20030166197 and WO200032761. For planttranscription factors or transcriptional regulators of abiotic stress,see e.g. US20040098764 or US20040078852.

Other genes and transcription factors that affect plant growth andagronomic traits such as yield, flowering, plant growth and/or plantstructure, can be introduced or introgressed into plants, see e.g.WO97/49811 (LHY), WO98/56918 (ESD4), WO97/10339 and US6573430 (TFL),US6713663 (FT), WO96/14414 (CON), WO96/38560, WO01/21822 (VRN1),WO00/44918 (VRN2), WO99/49064 (GI), WO00/46358 (FRI), WO97/29123,US6794560, US6307126 (GAI), WO99/09174 (D8 and Rht), and WO2004076638and WO2004031349 (transcription factors).

TABLE 1 Yield data for 09N12CY in DM in T/A compared to other varietiesat multiple locations. Date Test Planted Syn Year No. LSD Location Mo/YrGen Harvested Cuts 09N12CY 55V48 55V12 54V09 .05 CV % Connell, 5/2009 12010 5 13.52 14.38 12.87 13.89 0.69 3.7 WA 5/2009 1 2011 4 12.97 9.7211.94 12.22 0.64 3.7 Arlington, 5/2009 1 2010 4 9.17 13.05 8.83 9.760.38 3.0 WI 5/2009 1 2011 3 6.04 6.26 5.87 6.14 0.39 4.6 Connell, WA4/2010 2 2011 4 12.92 13.65 12.40 13.01 0.82 3.4 Moses Lake, 4/2010 22011 4 10.94 11.22 10.57 10.33 0.82 4.4 WA Rosemont, 4/2010 2 2011 44.74 5.00 5.18 5.14 0.42 6.5 MN East 4/2010 2 2011 4 6.28 5.91 6.88 6.110.92 5.1 Lansing, Ml Landisville, 4/2010 2 2011 4 9.77 9.76 9.19 9.361.16 5.7 PA Platteville, 4/2010 2 2011 4 6.35 6.70 6.15 6.13 0.31 10.4WI Arlington, 4/2010 2 2011 4 6.06 6.48 5.28 5.67 0.54 8.9 WI

TABLE 2 Mean Annual Yield in Tons DM/Acre. # of Total Years # of VarietyHar- Har- Name vested vests 09N12CY 55V48 55V12 54V09 09N12CY 11 44 8.9855V48 11 44 8.98 9.28 55V12 11 44 8.98 8.65 54V09 11 44 8.98 8.89

TABLE 3 Persistence data for 09N12CY compared to other varieties(Percent of stand). No. of Date of Date of Date Years No. of ReadingsReadings Test Syn Seeded Har- Har- Initial Final 09N12CY 09N12CY 55V1255V12 53H92 53H92 LSD CV Location Gen (Mo/Yr) vested vests (Mo/Yr)(Mo/Yr) Initial Final Initial Final Initial Final .05 % Arlington, 15/2009 3 9 7/09 10/11 100 99.7 100 98.6 100 96.9 4.1 2.5 WI

TABLE 4 Fall dormancy as determined from spaced plantings relative tothree standard check varieties. Test conducted by Pioneer Hi-BredInternational Inc., at Arlington, WI. Dormancy Date of Score or LastDate Average Syn Cut Measured LSD CV Variety FDC¹ Height Gen (Mo/Yr)(Mo/Yr) .05 % 09N12CY 29 2 9/11 10/11 1.5 5 Legend 4.0 24 Archer 5.0 30ABI 700 6.0 33

TABLE 5 Standability Expression (Lodging Resistance) Scores for 09N12CY.Test conducted by Pioneer Hi-Bred International Inc., at Arlington, WI.Kind of test conducted is Field. Lodging Year Unad- Ad- Stand- Resis-Test- Syn justed justed ability Variety tance ed Gen % R % R Index09N12CY R 2011 2 90 79 9.0 1. 54H11 R 77 68 7.7 2. 5454 S 10 9 1.0 3.Test Mean: 61 54 6.1 L.S.D. (.05%) 17 18 3.6 C.V. (%) 32 35

TABLE 6 Anthracnose (Race 1) Disease Scores for 09N12CY. Test conductedby Pioneer Hi-Bred International Inc., at Arlington, WI. Kind of testconducted is Greenhouse. Resis- Year Unad- Ad- tance Test- Syn justedjusted Variety Class ed Gen % R % R Score 09N12CY R 2010 2 46 53 1. ArcHR 61 70 2. Saranac AR R 3. Saranac S 4 5 4. Test Mean: 55 63 L.S.D.(.05%) 12 14 C.V. (%) 15 15

TABLE 7 Bacterial Wilt Disease Scores for 09N12CY. Test conducted byPioneer Hi-Bred International Inc., at Arlington, WI. Kind of testconducted is Greenhouse. Resis- Year Unad- Ad- tance Test- Syn justedjusted Variety Class ed Gen % R % R Score 09N12CY HR 2011 2 68 66 1.Vernal R 43 42 2. Narragansett S 3. Sonora S 3 3 4. Test Mean: 62 61L.S.D. (.05%) 12 12 C.V. (%) 10 10

TABLE 8 Fusarium Wilt Disease Scores for 09N12CY. Test conducted byPioneer Hi-Bred International Inc., at Arlington, WI. Kind of testconducted is Field. Resis- Year Unad- Ad- tance Test- Syn justed justedVariety Class ed Gen % R % R Score 09N12CY HR 2011 2 52 61 1. Agate HR46 54 (Field) Agate HR (Greenhouse) 2. Moapa 69 HR (Field) Moapa 69 HR(Greenhouse) 3. Narragansett MR (Field) Narragansett N/A (Greenhouse) 4.MNGN-1 S 4 5 Test Mean: 37 43 L.S.D. (.05%) 15 18 C.V. (%) 25 25

TABLE 9 Verticillium Wilt Disease Scores for 09N12CY. Test conducted byPioneer Hi-Bred International Inc., at Arlington, WI. Kind of testconducted is Greenhouse. Resis- Year Unad- Ad- tance Test- Syn justedjusted Variety Class ed Gen % R % R Score 09N12CY HR 2010 2 62 73 1.Vertus R 34 40 2. Oneida VR HR 3. Saranac S 2 2 4. Test Mean: 51 60L.S.D. (.05%) 21 25 C.V. (%) 34 34

TABLE 10 Phytophthora Root Rot Disease Scores for 09N12CY. Testconducted by Pioneer Hi-Bred International Inc., at Connell, WA. Kind oftest conducted is Seedling. Resis- Year Unad- Ad- tance Test- Syn justedjusted Variety Class ed Gen % R % R Score 09N12CY HR 2009 1 71 78 1.WAPH-1 HR 50 55 (seedling) 2. MNP-D1 R (seedling) 3. Agate R 4. SaranacS 0 0 Test Mean: 65 72 L.S.D. (.05%) 12 13 C.V. (%) 14 14

TABLE 11 Aphanomyces Root Rot (Race 1) Disease Scores for 09N12CY. Testconducted by Pioneer Hi-Bred International Inc., at Arlington, WI. Kindof test conducted is Greenhouse. Resis- Year Unad- Ad- tance Test- Synjusted justed Variety Class ed Gen % R % R Score 09N12CY HR 2010 2 5460 1. WAPH-1 (Race 1) R 45 50 2. WAPH-1 (Race 2) S 3. WAPH-5 (Race 2) R4. Saranac (Races S 4 4 1 & 2) Test Mean: 51 57 L.S.D. (.05%) 11 12 C.V.(%) 16 16

TABLE 12 Pea Aphid Insect Scores for Scores for 09N12CY. Test conductedby Pioneer Hi-Bred International Inc., at Arlington, WI. Kind of testconducted is Greenhouse. Resis- Year Unad- Ad- tance Test- Syn justedjusted Variety Class ed Gen % R % R Score 09N12CY R 2011 2 28 34 1.CUF-101 HR 2. PA-1 HR 3. Kanza R 4. Baker R 37 45 5. Caliverde S 6.Moapa 69 S 7. Vernal S 4 5 8. Ranger S Test Mean: 26 32 L.S.D. (.05%) 1012 C.V. (%) 27 27

TABLE 13 Spotted Alfalfa Aphid Insect Scores for 09N12CY. Test conductedby Pioneer Hi-Bred International Inc., at Connell, WA. Kind of testconducted is Greenhouse. Resis- Year Unad- Ad- tance Test- Syn justedjusted Variety Class ed Gen % R % R Score 09N12CY R 2009 1 29 35 1.CUF-101 HR 2. Baker R 41 50 3. Mesa Sirsa R 4. Kanza R 5. Caliverde S 6.Arc S 7. OK08 S 8. Ranger S 0 0 Test Mean: 36 44 L.S.D. (.05%) 11 13C.V. (%) 26 26

TABLE 14 Stem Nematode Scores for 09N12CY. Test conducted by PioneerHi-Bred International Inc., at Connell, WA. Kind of test conducted isControlled Environment, Greenhouse. Resis- Year Unad- Ad- tance Test-Syn justed justed Variety Class ed Gen % R % R Score 09N12CY R 2009 1 3540 1. Vernema HR 52 60 2. Lahontan R 3. Lew R 4. Ranger S 3 3 5. Moapa69 S Test Mean: 43 50 L.S.D. (.05%) 9 10 C.V. (%) 15 15

TABLE 15 Aphanomyces Root Rot (Race 2) Disease Scores for 09N12CY. Testconducted by Pioneer Hi-Bred International Inc., at Arlington, WI. Kindof test conducted is Lab. Resis- Year Unad- Ad- tance Test- Syn justedjusted Variety Class ed Gen % R % R Score 09N12CY R 2011 2 33 33 1.WAPH-1 (Race 1) R 2. WAPH-1 (Race 2) S 3. WAPH-5 (Race 2) R 50 50 4.Saranac (Races S 0 0 1 & 2) Test Mean: 22 22 L.S.D. (.05%) 9 9 C.V. (%)29 29

All disease tests conducted for National Alfalfa and MiscellanceousLegume Variety Review Board for AOSCA certification and were conductedby standard procedures and scoring systems as described in the NAAICStandard Tests to Characterize Alfalfa Cultivars, maintained online onthe NAAIC's website.

It is understood that the above description is intended to beillustrative, and not restrictive. Many other embodiments will beapparent to those of skill in the art upon reviewing the abovedescription. The scope of the invention should, therefore, be determinedwith reference to the appended claims, along with the full scope ofequivalents to which such claims are entitled.

DEPOSITS

Applicant will make a deposit of at least 2500 seeds of Alfalfa Variety09N12CY with the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va. 20110-2209, USA, with ATCC DepositNo. ______. The seeds to be deposited with the ATCC on ______ will beobtained from the seed of the variety maintained by Pioneer Hi-BredInternational, Inc., 7250 NW 62nd Avenue, Johnston, Iowa, 50131 sinceprior to the filing date of this application. Access to this seed willbe available during the pendency of the application to the Commissionerof Patents and Trademarks and persons determined by the Commissioner tobe entitled thereto upon request. Upon allowance of any claims in theapplication, the Applicant will make the deposit available to the publicpursuant to 37 C.F.R. §1.808. This deposit of the Alfalfa Variety09N12CY will be maintained in the ATCC depository, which is a publicdepository, for a period of 30 years, or 5 years after the most recentrequest, or for the enforceable life of the patent, whichever is longer,and will be replaced if it becomes nonviable during that period.Additionally, Applicant has or will satisfy all of the requirements of37 C.F.R. §§1.801-1.809, including providing an indication of theviability of the sample upon deposit. Applicant has no authority towaive any restrictions imposed by law on the transfer of biologicalmaterial or its transportation in commerce. Applicant does not waive anyinfringement of rights granted under this patent or under the PlantVariety Protection Act (7 USC 2321 et seq.).

We claim:
 1. Seed of an Alfalfa Variety 09N12CY, representative seed having been deposited under ATCC Accession Number ______.
 2. An alfalfa plant, or a part thereof, produced by growing the seed of claim
 1. 3. Pollen of the plant of claim
 2. 4. An ovule from the plant of claim
 2. 5. A tissue culture of regenerable cells or regenerable protoplasts from the plant of claim
 2. 6. A tissue culture according to claim 5, wherein a cell or protoplast of the tissue culture is derived from a tissue or cell selected from the group consisting of leaves, roots, root tips, root hairs, anthers, pistils, stamens, pollen, ovules, flowers, seeds, embryos, stems, buds, cotyledons, hypocotyls, cells and protoplasts.
 7. An alfalfa plant regenerated from the tissue culture of claim 5, wherein the regenerated plant has all of the morphological and physiological characteristics of alfalfa variety 09N12CY, representative seed of said alfalfa variety having been deposited under ATCC Accession Number ______.
 8. A process for producing a first generation progeny alfalfa seed comprising crossing a first parent alfalfa plant with a second parent alfalfa plant and harvesting the resultant alfalfa seed, wherein said first parent alfalfa plant or said second parent alfalfa plant is the alfalfa plant of claim
 2. 9. Growing the seed of claim
 1. 10. A process for transforming the plant or plant part of claim 2 with a transgene.
 11. The seed of claim 1 further comprising a transgene.
 12. The seed of claim 11 wherein the transgene confers a trait selected from the group consisting of herbicide resistance, insect resistance, disease resistance, improved digestibility, improved energy content, male sterility, and improved winterhardiness.
 13. Producing another synthetic variety wherein the seed of claim 1 is combined with seed of one or more other alfalfa plants.
 14. A process for producing alfalfa seed by growing the plant of claim 2 and allowing the plant of claim 2 to cross pollinate with one or more different alfalfa plants.
 15. Harvesting the seed of claim
 2. 